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. 2008 Oct 6;183(1):101–116. doi: 10.1083/jcb.200801099

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© 2008 Degtyarev et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 4. — Lysosomotropic agents accelerated cell death in combination with Akt KD. (A) CQ treatment caused accumulation of GFP-LC3 dots in Dox-treated PC3-shAkt123 cells. PC3-shAkt123 cells stably expressing GFP-LC3 were pretreated with or without 1 μg/ml Dox for 6 d and treated with or without 10 μM CQ. GFP fluorescence was imaged after 1 d of CQ treatment. Arrowheads point to representative GFP dots or clumps. Bar, 10 μm. (B) Effect of shAkt123 and 10 μM CQ on LC3 processing, PARP cleavage, and total Akt in PC3-shAkt123 cells treated with or without Dox or CQ. The ratios of LC3-II to LC3-I and cleaved (Cl) to full-length (FL) PARP were quantified from immunoblots of cell lysates made at days 1 and 2 of CQ treatment. Immunoblots of day 2 samples are shown. Molecular masses are indicated in kilodaltons parenthetically next to each protein. Data are representative of three independent experiments. (C) CQ promoted cell death in PC3 cells induced to express shAkt123, whereas 3-MA pretreatment delayed this effect. PC3-shAkt123 cells were preincubated with 1 μg/ml Dox and/or 1 mM 3-MA for 3 d before 10 μM CQ or 2.5 nM Ba was added. Cell viability was determined at days 2, 3, and 4 under 0.5% (C) or 0% (D) FBS after CQ or Ba was added. The percentage of the annexin V–positive PI-negative population was determined at days 2, 3, and 4 under 0.5% FBS. Caspase-3/7 activity was determined at days 2 and 3 under 0% FBS and expressed as relative fluorescence units (RFU, in thousands) normalized to the same number of cells. Error bars represent SD of three independent experiments.